1. Field of the Invention
The present invention relates to the purification of proteins, and, more particularly, to the purification of clotting Factor VIII:C by means of affinity chromatography.
2. Related Art
It is known that the clotting of human blood is a complicated process, involving a series of reactions mediated by 13 different factors. The cause of hemophilia A is the inability of the afflicted individual to synthesize one of these factors, known variously as antihemophilic factor, AHF, AHG, Factor VIII or Factor VIII:C, in amounts sufficient to support adequate clotting. About 40 percent of hemophiliac have no ability to synthesize AHF, while the others have diminished ability. Dried preparations of AHF concentrate are sold commercially for administration to hemophiliacs for treatment of bleeding or in advance of surgery. The AHF concentrate is obtained from plasma from human donors, through the use of known techniques. At the time of use, the dried concentrate is dissolved in sterile water, and the resulting solution is administered intravenously.
AHF concentrate contains a Factor VIII complex which consists of at least two components, one of which is called Factor VIII:C, in which C means that this component is responsible for the coagulation activity of the complex in the reaction chain. This component is considered to contain the antigen, as shown by means of antibodies, which are developed in certain persons suffering from hemophilia and which prevent the coagulation activity of Factor VIII:C. The antigen is called F.VIII:CAG. The other component has been called Factor VIII:RAG or F.VIII-related antigen. This antigen is different from the antigen F.VIII:CAG. The Factor VIII:C and the (antigen) Factor VIII:CAG are lacking in hemophilia of type A in a serious form. In this disease there is a normal content of Factor VIII:RAG. In von Willebrand's disease, there is a lack of Factor VIII:RAG in the blood and a corresponding lack of Factor VIII:C. For persons suffering from a serious form of von Willebrand's disease the lack of Factor VIII:RAG is almost total and the content of Factor VIII:C is about 5% of the normal content. In von Willebrand's disease, the activity of the so-called Factor VIII:RCF is highly reduced.
In November, 1984, the cloning of Factor VIII:C was reported by two groups. (Vehar et al., Nature, 312:337-342 (1984); Gitschier et al., Nature, 312:326-330 (1984); Toole, et al., Nature, 312:342-347 (1984)).
Both groups used similar methods for the DNA cloning and expression of Factor VIII:C. Oligonucleotide probes were synthesized on the basis of small sections of the amino acid sequence of purified Factor VIII:C (either porcine or human in origin), and used to screen an appropriate library of genomic clones to identify part of the Factor VIII:C gene. The gene itself turned out to be extraordinarily long.
For expression of protein, it was necessary to isolate cDNA clones. These were obtained either from a human T-cell hybridoma line (Genentech) or from human liver (Genetics Institute) and sequence analyses allowed the derivation of the entire 2,332 amino acid sequences of the mature protein. Both groups note that the protein is highly glycosylated and has an obvious domain structure with predictable homology to another clotting factor, Factor V, but also an unpredicted homology to ceruloplasmin--a serum protein believed to be involved in copper ion transport in the blood. The protein sequence also allows the definition of the main sites that are cleaved by thrombin in the process of activation of Factor VIII:C.
The expression of Factor VIII:C in mammalian cells required the reconstruction of a full length cDNA clone and its attachment to a viral promoter sequence. When this construction was introduced into either a hamster kidney cell (BHK) (Genentech) or a monkey kidney cell line (COS) (Genetics Institute), a human Factor VIII:C-like activity was secreted into the media in which the cells were grown. The concentrations of Factor VIII:C-like activity in the media were about one percent (Genetics Institute) or seven percent (Genentech) of the normal plasma concentration as assayed by a sensitive biochemical test based on the activation of Factor X and hydrolysis of a chromogenic substrate. Factor VIII activity is measured either by the chromogenic assay as the coagulation assay. In both instances, a WHO-accepted reference standard is used, and the results are reported in international units (IU) wherein 1 unit represents the Factor VIII activity in 1 ml of normal plasma. One mg of pure F.VIII is estimated to correspond to approximately 4500 IU. Both groups showed that the secreted activity (further purified by affinity chromatography at Genentech) was able to correct the clotting time of plasma from a hemophiliac. Evidence that these assays are indeed a measure of Factor VIII:C and not of some other clotting factor or non-specific activity, was provided by additional tests carried out by both groups.
The above-described cloning at Genentech is also described in EPO No. 0 160 457.
The purification described in Nature 312:337-338, supra, involved fractionation of proteins by TSK 4000 HPLC, and HPLC chromatography. Since these were analytical techniques, there is no discussion in the reference of the purities obtained by these techniques.
The cloning and expression of recombinant Factor VIII has subsequently been reported by other researchers. For example, Sarver et al., DNA 6(6):553-564 (1987), report purification of rF.VIII using an immobilized monoclonal antibody to VIII:C.
However, published information on the purification of rF.VIII is, at best, limited.
Various chromatography purification techniques have also been applied to plasma-derived Factor VIII.
Zimmerman et al., Re. No. 32,011, disclose a method of preparing high purity Factor VIII either from a commercially available Factor VIII concentrate or from porcine plasma. The process involves, as the first step, the immunoadsorption of F.VIII/vWF onto murine, a monoclonal antibody specific for vWF. The Factor VIII is then eluted with a calcium chloride solution (0.01M to 0.03M) and then concentrated with an aminohexyl agarose column.
The Factor VIII thus produced is described as having a specific activity of around 2300 when prepared from a Factor concentrate and being free substantially of von Willebrand Factor.
Various other methods using affinity chromatography have been described for concentrating Factor VIII from plasma. Andersson, EP No. 197901, discloses a method for preparing fragments of AHF using immunoaffinity chromatography followed by HPLC on an anion-exchange adsorbent. The anion exchange adsorbent may be Mono Q gel or TSK DEAE 5 PW gel. Fragments are then obtained by incubation with thrombin.
Johnson, U.S. Pat. No. 4,397,841, discloses preparation of Factor VIII:C by fractionation of plasma with a sequence of adsorption steps employing polyelectrolyte copolymers in the presence of heparin. A suitable resin is a copolymer of ethylene and maleic anhydride.
Other methods using affinity techniques in the downstream processing of plasma-derived and recombinant proteins are discussed in Lowe, J. Biotechnology, 1:3-12 (1984). None of the methods discussed use lectins.
Lectins have been hitherto used in the analysis of carbohydrate portions of glycoproteins. Kornfeld et al., J. Biol. Chem., 256:6633-6640 (1981), disclose the carbohydrate-binding specificity of pea and lentil lectins. Labelled human IgG immunoglobulins were passed over various lectin-Sepharose columns. The fractionation pattern was then compared to the known glycosylation pattern.
In J. Biol. Chem., 257:11230-11234 (1982), Cummings et al. report that various hemaglutinin lectins can be used to distinguish various Asn-linked oliogosaccharides with different branching patterns. This was used to separate glycopeptides prepared from bovine thyroglobulin.
The differentiation of ribonucleases from different human organs has also been studied by the fractionation of various ribonuclease preparations on lectin-affinity columns. Each lectin column used showed a different binding pattern for each ribonuclease studied, demonstrating that the various ribonucleases have different glycosylation patterns.
This review of the prior art suggests that lectin chromatography has not been previously used in the recovery or isolation of proteins, whether plasma-derived or of recombinant origin.